Purification and characterization of 4-methylmuconolactone methylisomerase, a novel enzyme of the modified 3-oxoadipate pathway in the gram-negative bacterium Alcaligenes eutrophus JMP 134.

4-Carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone) isomerase, transforming 4-methyl-2-enelactone to 3-methyl-2-enelactone, was purified from a derivative strain of Pseudomonas sp. B13, named B13 FR1, carrying the plasmid pFRC2OP. This plasmid contained the isomerase gene cloned from Alcaligenes eutrophus JMP 134, which uses 4-methyl-2-enelactone as a carbon source. The enzyme consists of a single peptide chain of Mr 40,000 as judged by SDS/PAGE. In addition to 4-methyl-2-enelactone, the putative reaction intermediate, 1-methyl-3,7-dioxo-2,6-dioxy-bicyclo[3.3.0]octane (1-methylbislactone), was a substrate for the enzyme, but kinetic data presented did not favour its role as a reaction intermediate. Isomeric methyl-substituted 4-carboxymethylbut-2-en-4-olides were neither substrates nor inhibitors. Possible reaction mechanisms are discussed.     

β-Methylmuconolactone, a key intermediate in the dissimilation of methylaromatic compounds by a modified 3-oxoadipate pathway evolved in nocardioform actinomycetes

Abstract p -Toluate-grown cells of Rhodococcus ruber N75, R. corallinus N657, R. rhodochrous N5 and Rhodococcus strains BCN1, BCN2 and 4PH1 metabolized 4-methylcatechol by a modified 3-oxoadipate pathway. Steps in the conversion of this compound to 4-methyl-3-oxoadipic acid were investigated. The conversion of 4-carboxymethyl-3-methylbut-2-en-1, 4-olide to 4-carboxymethyl-3-methylbut-2-en-1, 4-olide by a new enzyme is described.     

Purification and characterization of proline-beta-naphthylamidase, a novel enzyme from pig intestinal mucosa

An enzyme hydrolyzing proline-beta-naphthylamide was purified to apparent homogeneity from porcine intestinal mucosa. The purified enzyme appears to consist of three identical subunit polypeptides with a molecular weight of about 58,000 each, associated noncovalently. The enzyme is a glycoprotein, and the subunit polypeptide contains 3 residues each of mannose and N-acetylglucosamine. A wide variety of peptidase substrates were tested for the enzyme, and the results showed that it hydrolyzes only aminopeptidase substrates, such as proline-beta-naphthylamide, glycine-beta-naphthylamide, leucine-beta-naphthylamide, and alanine-beta-naphthylamide. Among these substrates, proline-beta-naphthylamide is most efficiently hydrolyzed as judged by the kcat/Km value. The optimum pH for this substrate is around 9. The enzyme also hydrolyzes efficiently the ester substrates of these amino acids. No hydrolytic activity was observed for the peptide and protein substrates tested. The proline-beta-naphthylamidase activity was drastically inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, indicating that the enzyme is a serine hydrolase, whereas it was slightly inhibited by aminopeptidase inhibitors, such as amastatin, bestatin, and puromycin. No significant homology was found for the NH2-terminal sequence of 27 amino acid residues with any known protein sequences. From these results we conclude that the enzyme is a protein which has not been described before.     

Purification and characterization of a novel enzyme, arylalkyl acylamidase, from Pseudomonas putida Sc2.

A novel enzyme, arylalkyl acylamidase, which shows a strict specificity for N-acetyl arylalkylamines, but not acetanilide derivatives, was purified from the culture broth of Pseudomonas putida Sc2. The purified enzyme appeared to be homogeneous, as judged by native and SDS/PAGE. The enzyme has a molecular mass of approximately 150 kDa and consists of four identical subunits. The purified enzyme catalyzed the hydrolysis of N-acetyl-2-phenylethylamine to 2-phenylethylamine and acetic acid at the rate of 6.25 mumol.min-1.mg-1 at 30 degrees C. It also catalyzed the hydrolysis of various N-acetyl arylalkylamines containing a benzene or indole ring, and acetic acid arylalkyl esters. The enzyme did not hydrolyze acetanilide, N-acetyl aliphatic amines, N-acetyl amino acids, N-acetyl amino sugars or acylthiocholine. The apparent Km for N-acetylbenzylamine, N-acetyl-2-phenylethylamine and N-acetyl-3-phenylpropylamine are 41 mM, 0.31 mM and 1.6 mM, respectively. The purified enzyme was sensitive to thiol reagents such as Ag2SO4, HgCl2 and p-chloromercuribenzoic acid, and its activity was enhanced by divalent metal ions such as Zn2+, Mg2+ and Mn2+.     

Purification and characterization of 3-dehydroshikimate dehydratase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa.

3-Dehydroshikimate dehydratase catalyzes the third reaction in the inducible quinic acid catabolic pathway of Neurospora crassa and is encoded in the qa-4 gene of the qa gene cluster. As part of continuing genetic and biochemical studies concerning the organization and regulation of this gene cluster, 3-dehydroshikimate dehydratase has been purified and characterized biochemically. The enzyme was purified 1650-fold using the following techniques: 1) (NH4)2SO4 fractionation; 2) ion exchange chromatography on DEAE-cellulose; 3) gel filtration on Sephadex G-100; 4) ion exchange chromatography on Cellex QAE (quaternary aminoethyl); and 5) hydroxylapatite chromatography. 3-Dehydroshikimate dehydratase is a monomer with a molecular weight of about 37,000 and a sedimentation coefficient of 3.27 S. It has a Km value of 5.9 X 10(-4) and an average isoelectric point of 4.92. The purified enzyme is extremely sensitive to thermal denaturation but can be significantly stabilized by Mg2+ ions. The purified enzyme also exhibits maximal catalytic activity only when assayed in the presence of certain divalent cations, e.g. magnesium. The NH2-terminal residue of 3-dehydroshikimate dehydratase is proline, and its alpha-amino group is unblocked.     

Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme

A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min^–1 (mg protein)^–1. The dismutase obeyed Michaelis-Menten kinetics. The V _max and K _m calculated for chlorite were 2,200 U (mg protein)^–1 and 170 μM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole. Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase displayed maxima for activity at pH 6.0 and 30° C. Received: 9 April 1996 / Accepted: 12 August 1996     

Purification,overproduction, and partial characterization of beta-RFAP synthase,a key enzyme in the methanopterin biosynthesis pathway

Methanopterin is a folate analog involved in the C1 metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria. Although a pathway for methanopterin biosynthesis has been described in methanogens, little is known about the enzymes and genes involved in the biosynthetic pathway. The enzyme beta-ribofuranosylaminobenzene 5'-phosphate synthase (beta-RFAP synthase) catalyzes the first unique step to be identified in the pathway of methanopterin biosynthesis, namely, the condensation of p-aminobenzoic acid with phosphoribosylpyrophosphate to form beta-RFAP, CO2, and inorganic pyrophosphate. The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding beta-RFAP synthase has not yet been identified. In the present work, we report on the purification to homogeneity of beta-RFAP synthase. The enzyme was purified from the methane-producing archaeon Methanosarcina thermophila, and the N-terminal sequence of the protein was used to identify corresponding genes from several archaea, including the methanogen Methanococcus jannaschii and the sulfate-reducing archaeon Archaeoglobus fulgidus. The putative beta-RFAP synthase gene from A. fulgidus was expressed in Escherichia coli, and the enzymatic activity of the recombinant gene product was verified. A BLAST search using the deduced amino acid sequence of the beta-RFAP synthase gene identified homologs in additional archaea and in a gene cluster required for C1 metabolism by the bacterium Methylobacterium extorquens. The identification of a gene encoding a potential beta-RFAP synthase in M. extorquens is the first report of a putative methanopterin biosynthetic gene found in the Bacteria and provides evidence that the pathways of methanopterin biosynthesis in Bacteria and Archaea are similar.     

Purification and characterization of a novel enzyme, N-carbamoylsarcosine amidohydrolase, from Pseudomonas putida 77

N-Carbamoylsarcosine amidohydrolase, a novel enzyme involved in the microbial degradation of creatinine in Pseudomonas putida 77, was purified 27-fold to homogeneity with a 63% overall recovery through simple purification procedures including successive ammonium sulfate fractionation, DEAE-cellulose chromatography, and crystallization. The relative molecular mass of the native enzyme estimated by the ultracentrifugal equilibrium method is 102,000 +/- 5000, and the subunit Mr is 27,000. The Km and Vm values for N-carbamoylsarcosine are 3.2 mM and 1.75 units/mg protein, respectively. Ammonia, carbon dioxide, and sarcosine were formed stoichiometrically from N-carbamoylsarcosine through the action of the purified enzyme preparation. N-Carbamoyl amino acids with a methyl group or hydrogen atom on the amino-N atom and possessing glycine, D-alanine, or one of their derivatives as an amino acid moiety served well as substrates for N-carbamoylsarcosine amidohydrolase. N-Carbamoylsarcosine, N-methyl-N-carbamoyl-D-alanine, N-carbamoylglycine, and N-carbamoyl-D-alanine were hydrolyzed at relative rates of 100, 12.8, 9.8, and 7.3, respectively, by the enzyme. N-Carbamoyl derivatives of D-tryptophan, D-phenylalanine, and those of some other amino acids including D-phenylglycine and p-hydroxy-D-phenylglycine were also hydrolyzed by the enzyme. For the L-isomers of all N-carbamoyl amino acids tested there was no production of ammonia, carbon dioxide, or the corresponding amino acids due to the action of the enzyme. Cupric, mercuric, and silver ions inhibited the enzyme strongly, and some thiol reagents were also found to be inhibitory.     

Purification, kinetic characterization, and molecular cloning of a novel enzyme, ecdysteroid 22-kinase

This is the first report succeeding in the isolation and characterization of an enzyme and its gene involved in the phosphorylation of a steroid hormone. It has been demonstrated that ecdysteroid 22-phosphates in insect ovaries, which are physiologically inactive, serve as a "reservoir" that supplies active free ecdysteroids during early embryonic development and that their dephosphorylation is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (Yamada, R., and Sonobe, H. (2003), J. Biol. Chem. 278, 26365-26373). In this study, ecdysteroid 22-kinase (EcKinase) was purified from the cytosol of the silkworm Bombyx mori ovaries to about 1,800-fold homogeneity in six steps of column chromatography and biochemically characterized. Results obtained indicated that the reciprocal conversion of free ecdysteroids and ecdysteroid 22-phosphates by two enzymes, EcKinase and ecdysteroid-phosphate phosphatase, plays an important role in ecdysteroid economy of the ovary-egg system of B. mori. On the basis of the partial amino acid sequence obtained from purified EcKinase, the nucleotide sequence of the cDNA encoding EcKinase was determined. The full-length cDNA of EcKinase was composed of 1,850 bp with an open reading frame encoding a protein of 386 amino acid residues. The cloned cDNA was confirmed to encode the functional EcKinase using the transformant harboring the open reading frame of EcKinase. A data base search showed that EcKinase has an amino acid sequence characteristic of phosphotransferases, in that it harbors Brenner's motif and putative ATP binding sites, but there are no functional proteins that share high identity with the amino acid sequence of EcKinase.     

Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12

A novel fibrinolytic enzyme from Rhizopus chinensis 12 was purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange, and gel filtration chromatography. The purification protocol resulted in a 893-fold purification of the enzyme, with a final yield of 42.6%. The apparent molecular weight of the enzyme was 18.0 kDa, determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 16.6 kDa by gel filtration chromatography, which revealed a monomeric form of the enzyme. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5±0.1. The enzyme hydrolyzed fibrin. It cleaved the , , and chains of fibrinogen simultaneously, and it also hydrolyzed casein and N-succinyl-Ala-Ala-Pro-Phe-pNA. The enzyme had an optimal temperature of 45°C, and an optimal pH of 10.5. EDTA, PCMB, and PMSF inhibited the activity of the enzyme, and SBTI, Lys, TPCK, and Aprotinine had no obvious inhibition, which suggested that the activity center of the enzyme had hydrosulfuryl and metal. The first 12 amino acids of the N-terminal sequence of the enzyme were S-V-S-E-I-Q-L-M-H-N-L-G and had no homology with that of other fibrinolytic enzyme from other microbes.     

Purification and characterization of an exo-beta-D-glucosaminidase, a novel type of enzyme, from Nocardia orientalis

A new enzyme capable of hydrolyzing chitobiose, which is an induced enzyme, was purified to apparent homogeneity from the culture filtrate of Nocardia orientalis IFO 12806. Biospecific affinity chromatography on chitotriitol-Sepharose CL-4B was effective for purification of this enzyme. It is clearly demonstrated that the enzyme is an exo-hydrolase, removing single glucosamine residues from the nonreducing terminal of a sequence of beta-(1----4)-linked glucosamine chain, such as chitosan and chitooligosaccharides, and therefore characterized as an exo-beta-D-glucosaminidase. The enzyme was found to show maximum activity on chitotetraose, chitopentaose, and their corresponding alcohols and a slight decrease in rate on longer chain lengths of substrates. A significant decrease in rate was observed using p-nitrophenyl beta-D-glucosaminide and chitobiitol as substrates. In the hydrolysis of partially acetylated chitosans, the enzyme appeared to be effective in cleaving glucosamine from the GlcN beta 1----4GlcNAc beta 1----sequence as well as the GlcN beta 1----4GlcN beta 1----sequence. These observations suggest that the second residue from the terminal plays an important role in enzyme activity, but the enzyme permits the replacement of glucosamine at the second residue by N-acetylglucosamine.     

Purification and characterization of a novel extracellular Streptomyces lividans 66 enzyme inactivating fusidic acid.

The wild-type strain Streptomyces lividans 66 is resistant against the steroid-like antibiotic fusidic acid. Comparative studies of the wild-type strain and a fusidic acid-sensitive mutant allowed the identification of an extracellular enzyme which inactivates fusidic acid. With the help of a combination of ultrafiltration and chromatographies with Phenyl-Sepharose and an anion exchanger, the enzyme was highly purified. Its apparent molecular mass is 48 kDa, its optimal activity ranges between 45 and 55 degrees C, and its optimal pH is 6.0 to 9.0. It is stimulated by neither monovalent nor divalent ions. The enzyme acts as a specific esterase which removes the acetyl group at C-16 from fusidic acid. The resulting intermediate is unstable, and spontaneous lactonization between C-21 and C-16 occurs rapidly.     

Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme.

An egg-specific NADase has been purified to homogeneity from the ovotestis of the opisthobranch mollusk Aplysia californica. Unlike other NADases, the Aplysia enzyme generates primarily cyclic-ADP-ribose (cADPR) rather than ADP-ribose from NAD. cADPR has been shown to stimulate the release of Ca2+ from microsomes prepared from sea urchin egg and, when injected into intact eggs, to activate the cortical reaction, multiple nuclear cycles, and DNA synthesis. The Aplysia enzyme was initially identified as an inhibitor of cholera and pertussis toxin-catalyzed ADP-ribosylation. By the use of an NADase assay, it was purified from the aqueous-soluble fraction of ovotestis by sequential column chromatography. The enzyme has an apparent molecular mass of 29 kDa, a Km for NAD of 0.7 mM, and a turnover rate of approximately 27,000 mol NAD.min-1.mol enzyme-1 at 30 degrees C. Monoclonal antibodies were generated to the NADase. Immunoblots of two-dimensional gels revealed multiple isoforms of the enzyme, with pls ranging from 8.1 to 9.8. The multiple isoforms were resolved with a cation exchange high-pressure liquid chromatography column and shown to generate cADPR. Immunohistochemical analysis of cryostat sections of Aplysia ovotestis shows that the enzyme is specific to the eggs and restricted to large 5- to 10-microns granules or vesicles. To date the cADPR-generating enzyme activity has been identified in various organisms, including mammals. The Aplysia enzyme is the first example in which the enzyme that generates cADPR has been purified. All of the available evidence indicates that this NADase is a second-messenger enzyme, implying that other NADases may serve a similar function.     

Purification and characterization of a novel subtype a3 botulinum neurotoxin

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are of considerable importance due to their being the cause of human and animal botulism, their potential as bioterrorism agents, and their utility as important pharmaceuticals. Type A is prominent due to its high toxicity and long duration of action. Five subtypes of type A BoNT are currently recognized; BoNT/A1, -/A2, and -/A5 have been purified, and their properties have been studied. BoNT/A3 is intriguing because it is not effectively neutralized by polyclonal anti-BoNT/A1 antibodies, and thus, it may potentially replace BoNT/A1 for patients who have become refractive to treatment with BoNT/A1 due to antibody formation or other modes of resistance. Purification of BoNT/A3 has been challenging because of its low levels of production in culture and the need for innovative purification procedures. In this study, modified Mueller-Miller medium was used in place of traditional toxin production medium (TPM) to culture C. botulinum A3 (CDC strain) and boost toxin production. BoNT/A3 titers were at least 10-fold higher than those produced in TPM. A purification method was developed to obtain greater than 95% pure BoNT/A3. The specific toxicity of BoNT/A3 as determined by mouse bioassay was 5.8 × 10(7) 50% lethal doses (LD(50))/mg. Neutralization of BoNT/A3 toxicity by a polyclonal anti-BoNT/A1 antibody was approximately 10-fold less than the neutralization of BoNT/A1 toxicity. In addition, differences in symptoms were observed between mice that were injected with BoNT/A3 and those that were injected with BoNT/A1. These results indicate that BoNT/A3 has novel biochemical and pharmacological properties compared to those of other subtype A toxins.     

Purification and characterization of a novel mycolic acid exchange enzyme from Mycobacterium smegmatis

We have isolated and purified to homogeneity an alpha,alpha'-trehalose 6-monomycolate:alpha,alpha'-trehalose mycolyltransferase (trehalose mycolyltransferase) from Mycobacterium smegmatis that catalyzes the exchange of a mycolyl group between trehalose, trehalose 6-monomycolate (TM), and trehalose 6,6'-dimycolate (TD). This enzyme was prominent in M. smegmatis and it catalyzed the following reactions. TM + [14C]trehalose in equilibrium [14C]TM + trehalose [14C]TM + TM in equilibrium [14C]TD + trehalose This enzyme was purified by (i) ammonium sulfate fractionation, (ii) QAE-Sephadex A-50 column chromatography, (iii) gel filtration on a Sephadex G-75 column, and (iv) SP-Sephadex C-50 column chromatography. The purified protein yielded a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 25,000. This enzyme was a glycoprotein, had no cofactor requirement, and was highly specific for alpha,alpha'-trehalose as the mycolate acceptor. It was less specific for the acyl donor group since the palmitoyl group in trehalose 6-monopalmitate was easily exchangeable. There was no TM acylhydrolase activity in the purified enzyme, suggesting that it is probably associated with the anabolic pathway of mycolic acid metabolism. We postulate the formation of a mycolyl-enzyme intermediate in this reaction. Such an intermediate could play a central role in the transfer of mycolic acid to form the prominent cell wall components of mycobacterial TD and possibly murein-arabinogalactan-mycolate.     

Purification and characterization of a ketimine-reducing enzyme

An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named ketimines has been detected and purified 2500-fold from pig kidney. Some molecular and kinetic properties of this enzyme have been determined. The enzymatic reduction proceeds with a classical ping-pong mechanism and some results suggest that the true substrate has the ketiminic structure and is in equilibrium with the enaminic and keto-open forms. As previously described, ketimines arise from the deamination of a number of sulfur-containing amino acids, i.e. L-cystathionine, L-lanthionine and S-aminoethyl-L-cysteine, catalyzed by a widespread mammalian transaminase. The enzymatic reduction products of ketimines have been identified as cyclothionine, 1,4-thiomorpholine 3,5-dicarboxylic acid and 1,4-thiomorpholine 3-carboxylic acid. Some of these compounds have been detected in mammals, thus suggesting a possible role of this enzyme in their biosynthesis.     

Purification,biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262

Fibrinolytic proteases are enzymes that degrade fibrin. They provide a promising alternative to existing drugs for thrombolytic therapy. A protease isolated from the filamentous fungus Mucor subtilissimus UCP 1262 was purified in three steps by ammonium sulfate fractionation, ion exchange, and molecular exclusion chromatographies, and characterized biochemically and structurally. The purified protease exhibited a molecular mass of 20 kDa, an apparent isoelectric point of 4.94 and a secondary structure composed mainly of α-helices. Selectivity for N-succinyl-Ala–Ala–Pro–Phe-p-nitroanilide as substrate suggests that this enzyme is a chymotrypsin-like serine protease, whose activity was enhanced by the addition of Cu²⁺, Mg²⁺, and Fe²⁺. The enzyme showed a fibrinolytic activity of 22.53 U/mL at 40 °C and its contact with polyethylene glycol did not lead to any significant alteration of its secondary structure. This protein represents an important example of a novel fibrinolytic enzyme with potential use in the treatment of thromboembolic disorders such as strokes, pulmonary emboli, and deep vein thrombosis.

     

Purification and characterization of a novel phosphorus-oxidizing enzyme from Pseudomonas stutzeri WM88

The ptxD gene from Pseudomonas stutzeri WM88 encoding the novel phosphorus oxidizing enzyme NAD:phosphite oxidoreductase (trivial name phosphite dehydrogenase, PtxD) was cloned into an expression vector and overproduced in Escherichia coli. The heterologously produced enzyme is indistinguishable from the native enzyme based on mass spectrometry, amino-terminal sequencing, and specific activity analyses. Recombinant PtxD was purified to homogeneity via a two-step affinity protocol and characterized. The enzyme stoichiometrically produces NADH and phosphate from NAD and phosphite. The reverse reaction was not observed. Gel filtration analysis of the purified protein is consistent with PtxD acting as a homodimer. PtxD has a high affinity for its substrates with Km values of 53.1 +/- 6.7 microm and 54.6 +/- 6.7 microm, for phosphite and NAD, respectively. Vmax and kcat were determined to be 12.2 +/- 0.3 micromol x min(-1) x mg(-1) and 440 min(-1). NADP can substitute poorly for NAD; however, none of the numerous compounds examined were able to substitute for phosphite. Initial rate studies in the absence or presence of products and in the presence of the dead end inhibitor sulfite are most consistent with a sequential ordered mechanism for the PtxD reaction, with NAD binding first and NADH being released last. Amino acid sequence comparisons place PtxD as a new member of the d-2-hydroxyacid NAD-dependent dehydrogenases, the only one to have an inorganic substrate. To our knowledge, this is the first detailed biochemical study on an enzyme capable of direct oxidation of a reduced phosphorus compound.     

Purification and characterization of catabolic dehydroquinase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa.

Catabolic dehydroquinase which functions in the inducible quinic acid catabolic pathway in Neurospora crassa has been purified 8000-fold. The enzyme was purified by two methods. One used heat denaturation of contaminating proteins; the other used antibody affinity chromatography. The preparations obtained by these two methods were identical by all criteria. The purified enzyme is extremely resistant to thermal denaturation as well as denaturation 0y urea and guanidine hydrochloride at 25 degrees. It is irreversibly inactivated, although not efficiently dissociated, by sodium dodecyl sulfate and guanidine hydrochloride at 55 degrees. At pH 3.0, the enzyme is reversibly dissociated into inactive subunits. At high concentrations catabolic dehydroquinase aggregates into an inactive, high molecular weight complex. The native enzyme, which has a very high specific activity, has a molecular weight of approximately 220,000 and is composed of identical subunits of 8,000 to 12,000 molecular weight each. The native enzyme and the subunit are both asymmetric.